CDNA CLONING AND EXPRESSION OF POKEWEED ANTIVIRAL PROTEIN FROM SEEDS IN ESCHERICHIA-COLI AND ITS INHIBITION OF PROTEIN-SYNTHESIS IN-VITRO

Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development o f Phytolacca americana (pokeweed). We isolated, sequenced and characte rized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds, The cDNA of PAP-S consists of 1249 nucleo tides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%), It i s known from literature that PAP-S is more active in inhibiting protei n synthesis than other members of the PAP family. Therefore, the cDNA of PAP-S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified fro m spring leaves, In a rabbit translation system, the median inhibitory concentrations (IC50) Of recombinant PAP-S and native PAP were determ ined as 0.07 and 0.29 nM, respectively, Although the PAP-S protein in seeds is glycosylated, PAP-S can be expressed in Escherichia coli in a very active form, indicating that posttranslational modification in p okeweed does not seem to alter its ability to inhibit protein synthesi s. (C) 1997 Federation of European Biochemical Societies.